I try to optimize human BIII-tubulin and GAP43 primers, so I want to differentiate SH-SY5Y cells into cells with morphological and biochemical characteristics of mature neurons. Therefore, is it enough to differentiate SH-SY5Y cells with 10uM retinoic acid treatment for 7 days or further NGF, BDNF or RA treatment is required?
you can do it with RA alone. If its just for optimizing something RA is the easier cheaper way. Note, there will be some apoptosis with the differentiation. It might by worth plating less cells in your control population as they will keep dividing.
After 2 days RA treatment, you will already start to see some changes by eye. I would change the media and add fresh RA every 2-4 days depending on your set-up and weekends etc. RA is light sensitive so loosely cover with foil when the cells are in the incubator. Always treat cells after they are plated and adhered to the plastic.
Some people also use 5 uM or 1 uM RA if they want less aggressive effects. 10 uM is the absolute maximum I would go with these cells, but it is what I usually used.
Good luck with your research!
We used 1 uM ATRA for SHSY5Y cells and got decent neurite formation within a week For neurite formation, a combination of BMP-6 and ATRA1 uM worked best with IMR32 cells. Higher concentrations of RA were toxic as mentioned above. Hope this helps!
We tend to use 5uM RA in SH cells, which induces strong differentiation after 6 days.
We always stimulated SHSY5Y with 10 uM RA and after 6 days the cells expressed GAP43, MAP2 and neuronal-specific enolase.
Thank you all for suggestions. After 7 days of induction with 10 uM RA, I gat GAP43 expression.
What is the advantage and disadvantage for doing differentiation by just RA or RA combined with growth factor?
Thanks!
Thomas
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